Field of the Invention
The present invention relates to the field of genetic engineering, and more particularly relates to a method for enhancing N-acetylglucosamine production through glcK knockout of Bacillus subtilis. 
Description of the Related Art
N-acetylglucosamine (GlcNAc) is a monosaccharide which is widespread in bacteria, yeast, fungi, plants and animals. As a precursor of glycosaminoglycan building blocks, it plays an important role in cartilage and joint health in the human body. Therefore, GlcNAc has long been used as pharmaceuticals and dietary supplements to cure osteroarthritis and maintain cartilage and joint health. In addition, it has wide application in the cosmetic and pharmaceutical fields. Currently, GlcNAc is mainly produced through acid hydrolysis of chitin extracted from crab and shrimp shells; however, this extraction method poses problems such as severe environmental pollution and potential allergic reactions in consumers.
Bacillus subtilis (B. subtilis) has long been used as a production host for manufacturing pharmaceutically important biochemicals and industrially useful components that meet GRAS grade requirements. Therefore, constructing recombinant B. subtilis by metabolic engineering would be an effective strategy to produce GRAS grade GlcNAc.
In previous work, a recombinant B. subtilis named BSGN6-PxylA-glmS-P43-GNA1 was constructed to produce GlcNAc (Ref: Liu, Y. et al. Modular pathway engineering of Bacillus subtilis for improved N-acetylglucosamine production. Metab. Eng. 23: 42-52, 2014). And the fermentation medium for GlcNAc production was complex medium, which contained corn steep liquor, yeast extract, tryptone, glucose, etc. However, the stability and controllability of the fermentation process were severely influenced by the complex medium, due to the fact that the compositions of the complex medium in different batches were quite different, especially the corn steep liquor. In addition, the high cost of the complex medium has become a major constraint in industrial production of GlcNAc by recombinant B. subtilis. Thus, it would be a great strategy to use synthetic medium for fermentation, which would avoid the differences of the complex medium in different batches and significantly reduce the costs and facilitate the separation and purification of the product. However, experimental data showed that the growth of BSGN6-PxylA-glmS-P43-GNA1was slow and the yield of GlcNAc was significantly reduced in the synthetic medium compared to the complex medium. Therefore, it has become an urgent problem to enhance the GlcNAc production of recombinant B. subtilis in synthetic media.
The present invention provides a method to enhance the GlcNAc production of recombinant B. subtilis in synthetic media and provides other benefits as well.